This project proposes to study the mechanism whereby expression of the eucaryotic growth factor, platelet-derived growth factor (PDGF), results in oncogenic transformation. PDGF is a well-characterized mitogen for cells of mesenchymal origin. However, expression of one subunit of PDGF, as acquired by simian sarcoma virus, can result in oncogenic transformation. Four areas of research are proposed. (1) Structure-function studies: The sis oncogene, homologous to the PDGF-A gene, will be mutagenized to assess which portions of the protein are required for biological activity. These mutants will be assayed for transformation using retrovirus vectors, and their protein expression will be characterized. Deletion mutants will be constructed in the N-terminal and C-terminal coding regions. Point mutants will be constructed using oligonucleotide site-directed mutagenesis in the N-terminal signal sequence, and also in potential sites of glycosylation, disulfide formation, and post-translational proteolytic processing. (2) In vitro studies of sis gene: The phage SP6 promoter will be used to synthesize RNA in vitro, which will be translated using rabbit reticulocyte lysate. Tryptic peptide maps of the PDGF-related proteins sysnthesized in vitro will be determined, and compared with in vivo synthesized proteins. Coupled transcription/translation/membrane vesicle processing will be used to study signal sequence cleavage, glycosylation, and post-translational proteolytic processing. (3) Fusions of sis with membrane anchor sequences: A major issue is whether the sis gene product must be secreted to function in an autocrine fashion. Therefore, the PDGF coding region will be fused with typical membrane anchor sequences, e.g., from the vesicular stomatitis virus glycoprotein gene. The hybrid genes will be assayed for fibroblast transformation. The localization within the cell of the hybrid proteins will be determined. The hybrid proteins also will be assayed for interaction with the PDGF-receptor. (4) Cloning the gene encoding subunit B of PDGF: DNA probes derived form the sis gene have allowed cloning the cellular gene for the A subunit of PDGF, but not for the B subunit. Using oligonucleotide probes based on PDGF-B peptide sequences, the PDGF-B gene will be isolated from a genomic library and characterized. The cloned gene will be used to determine if PDGF-B expression results in fibroblast transformation by the sis gene, perhaps by formation of PDGF A:B heterodimers. (J)